The insulin-like growth factor 1 receptor (IGF1R) is an essential regulator of cell growth and transformation that promotes tumor cell proliferation, motility and protection from apoptosis. We have shown that in head and neck squamous cell carcinoma (HNSCC) cells IGF1R activation increases vascular endothelial growth factor (VEGF) synthesis and secretion, initiating an autocrine VEGF:VEGFR2 signaling loop. Our overall hypothesis stipulates that VEGF signaling via VEGFR2, leads to enhanced HNSCC tumorigenicity and invasive cell behavior. In strong support of this hypothesis we have identified a cluster of proteins involved in cell motility activated by VEGF stimulation. These proteins include human enhancer of filamentation1 (HEF1), cortactin, paxillin, and focal adhesion kinase. Of significance, HEF1 (or neural precursor cell expressed developmentally down-regulated 9/NEDD9) has been identified as a signature protein required for metastasis in melanoma and glioblastoma. Consistent with this, we have shown that VEGF stimulated migration, invasion and invadopodia formation are all dependent upon HEF1 expression. We will further define the role HEF1 plays in HNSCC invasive behavior in the following aims. The goal of AIM 1 is to define the mechanism through which HEF1 mediates VEGF induced HNSCC cell migration and invasion. Specifically, we will define the specific tyrosine residues in HEF1 that are phosphorylated in response to VEGFR2 activation using complementary biochemical techniques and site-directed mutagenesis. We will further define the effector(s) that bind to these sites of regulation and define the role of the N-terminal SH3 domain and its interacting proteins in invasion. In AIM 2 we will define VEGF regulation of invadopodia formation and the structural and functional contributions of HEF1 to this process. As a subset of this aim we will examine HEF1 localization to invadopodia, the role of its SH3 domain in this process and in matrix metalloproteinase delivery to invadopodia. In AIM 3 we will test the hypothesis that elevated HEF1 expression is prognostic for advanced stage HNSCC tumors in contributing to metastasis and underlying a poor prognosis. Five year survival studies will also be conducted. Human HNSCC tissue arrays and tumor/normal pairs will be examined for HEF1 expression and select activation of signaling pathways. We will also test the contribution of HEF1 to metastasis using mouse carcinogenesis and xenograft models. These studies will provide important evidence demonstrating a role for HEF1 in HNSCC metastatic signaling downstream of crosstalk to VEGFR2. This will lead to the development of novel strategies and therapeutic agents aimed at reducing the tumorigenic and metastatic effects of the IGF and VEGF pathways in HNSCC. PUBLIC HEALTH RELEVANCE: Cancer of the head and neck is the 6th most frequently occurring cancer worldwide, causing nearly 8,000 deaths per year nationally. Alcohol and tobacco are common risk factors, but additional factors contribute, including the IGF system and as shown here, VEGF signaling pathways. This proposal seeks to define the mechanisms by which VEGF receptors acting through the scaffold protein HEF1, regulate the invasive and metastatic behavior of head and neck squamous cell carcinoma.